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Regular Article |
a Dipartimento di Biologia Evolutiva e Comparata, Università di Napoli "Federico II," 80134 Napoli, Italy
ABSTRACT
The mechanisms of sperm adhesion and release within the mammalian oviduct are still poorly understood. In this in vitro study, a previously developed adhesion assay was used to analyze the effects of heparin, N-desulfated heparin, fucoidan, dextran sulfate, and dextran on bovine sperm-oviductal cell adhesion and release. Results showed that 1) all sulfated glycoconjugates were powerful inhibitors of sperm binding to oviductal monolayers in a dose-dependent manner, whereas N-desulfated heparin and dextran had no effect; 2) sperm pretreatment with heparin and fucoidan markedly inhibited adhesion; 3) treatment of oviductal monolayers with heparinase I, II, or sodium chlorate (an inhibitor of sulfation) had no effect on sperm adhesion; 4) sulfated glycoconjugates were also powerful and quick inducers of sperm release from oviductal monolayers; and 5) addition of sulfated glycoconjugates to the cocultures caused a sudden increase of bound-sperm flagellar beat frequencies, followed by a release of highly motile sperm. In conclusion, these data support the hypothesis that sulfated glycoconjugates may act as signals that induce sperm release and migration from the oviductal reservoir.
fallopian tubes, female reproductive tract, sperm, sperm capacitation/acrosome reaction
INTRODUCTION
The mammalian oviduct, the natural site of sperm storage and capacitation, is thought to provide a suitable environment that allows the maintenance of sperm fertilization competence until ovulation. Previous in vivo studies indicate that, after mating, sperm are sequestered in the lower oviduct [1], which acts as a functional sperm reservoir. Sperm fertile life span may be maintained by binding to epithelial cells lining the oviduct lumen [2, 3] and by specific oviduct secretions [4–8]. At the time of ovulation, after a still unknown signal, sperm may progress toward the upper oviduct for fertilization [9–11]. In particular, the ability of the isthmic epithelium to hold uncapacitated sperm remains unchanged before and during ovulation, suggesting that sperm release may be physiologically triggered by capacitation [11].
To dissect the mechanisms involved in the maintenance of sperm motility, viability, and fertile potential, different in vitro coculture systems have been developed. Positive effects of both oviductal secretions [4–8], conditioned media [12–15], specific oviduct glycoproteins [16], and binding to oviductal cells [14, 17–19] or even to isolated fractions of oviductal plasma membrane [20–22] have been demonstrated in vitro. Bound sperm have been shown to be progressively released under in vitro coculture conditions, and this process should mimic what occurs in vivo in response to a still unknown physiologic signal [14, 17].
Recently, we found that in vitro cultured oviductal cells selectively bind acrosome-intact sperm and retain this ability after sperm release, suggesting that capacitation changes play a key role in sperm release [23]. Because sperm adhesion and release from the oviductal reservoir represent two main steps to achieve a successful fertilization, a deep interest surrounds the research on molecules involved in such events. As regards sperm binding, carbohydrate recognition has been involved in a number of species [24–26], whereas heparin-like glycosaminoglycans, which normally are present in the oviductal fluid, have been regarded as potential in vivo capacitating agents, at least in the bovine species [8, 27]. Preliminary data on the role played by the sulfated glycoconjugates heparin and fucoidan [28] on bovine sperm-oviduct adhesion suggest that these molecules are able to directly modulate such an event. To test this hypothesis in the present paper, a previously developed quantitative assay for sperm binding [23] was used to analyze the effects of heparin and other sulfated and nonsulfated glycoconjugates on in vitro sperm-oviduct adhesion and release in the bovine species. Results demonstrate that sulfated glycoconjugates, and not their unsulfated counterparts, are powerful and quick inhibitors of cell adhesion as well as inducers of sperm release in vitro, causing the loss of sperm affinity for oviductal cells. Moreover, data suggest that their action is exerted directly on sperm through early capacitation effects. These findings support the hypothesis that changes of glycoconjugate concentration during the estrous cycle drive the release of fertilization-competent sperm from the oviductal reservoir, allowing their ascension toward the ampulla.
MATERIALS AND METHODS
Chemicals
The BSA (fraction V and fraction V fatty-acid free), heparin (sodium salt, purified from porcine intestinal mucosa; H3393), N-desulfated heparin (sodium salt, purified from porcine intestinal mucosa; D4776) heparinase I and II, fucoidan, dextran (average molecular weight [MW] of 500 000), dextran sulfate (prepared from dextran with an average MW of 500 000), Hoechst 33258 and 33342, and sodium chlorate were from Sigma (Milan, Italy). Percoll was from Amersham-Pharmacia (Milan, Italy). Fetal calf serum (FCS), gentamicin, fungizone, Hepes, and sodium bicarbonate were from Gibco (Milan, Italy). Glutaraldheyde was from Taab laboratories (Rome, Italy). Reagents and water for preparation of salines and culture media were all cell culture tested.
Oviductal Monolayers
Oviducts, including the uterotubal junction, isthmus, ampulla, and fimbria, were collected at the time of slaughter and transported to the laboratory in Dulbecco's PBS supplemented with 50 µg/ml of gentamicin at 4°C. Laminae of epithelial cells, recovered from oviducts of single animals by squeezing, were cultured in M199 supplemented with 50 µg/ml of gentamicin, 1 µg/ml of fungizone, and 10% w/v FCS as previously described [23]. Bovine oviductal epithelial cells (BOECs) were cultured in 6-cm Petri dishes (Falcon; Becton Dickinson, Milan, Italy) for 24–48 h and then transferred into four-well tissue culture dishes (Nunc, Milan, Italy) with 12-mm, glass, round coverslips on the well bottom. Fresh media changes were performed every 48 h. Cell confluence on glass surfaces was attained in approximately 7–10 days. Within each experiment, BOEC monolayers or swimming-everted vesicles, referred to as explants (and used within 24 h of culture), from a single individual were washed three times in modified Tyrode's albumin lactate pyruvate medium (sp-TALP) [27] and then left in this medium until sperm addition (1–3 h).
Sperm Preparation
The same batch of frozen bovine semen pooled from three bulls (0.5-ml straws, approximately 50 x 106 sperm per straw), obtained from Semen Italy (San Giuliano Saliceta, Modena, Italy), was used in all experiments. Straws were thawed in a water bath at 38°C for 30 sec and then washed and stained at the same time in a discontinuous Percoll gradient containing 10 µg/ml of Hoechst 33342. The semen samples were deposited on the top of the Percoll layers, which were prepared by depositing 1 ml of 90% w/v and 1 ml of 45% w/v Percoll to form two layers in a 15-ml centrifuge tube, and then centrifuged for 30 min at 200 x g. The supernatant was removed, and the pellet, which was resuspended in 300 µl of sp-TALP, was brought to 5 ml with sp-TALP, centrifuged at 200 x g for 10 min, and resuspended in 300 µl of sp-TALP.
Experimental Design
Oviduct selection was done on the basis of epithelial cell ciliary beating at the time of collection. Fully confluent oviductal monolayers or swimming-everted vesicles (i.e., explants) from single animals and a batch of frozen bovine semen pooled from three bulls were used in all experiments.
Within each experiment, oviductal monolayers or explants from one animal were incubated with the same sperm suspension at a final concentration of 0.2–1.0 x 106 motile sperm/well in 0.5 ml of sp-TALP into four-well tissue culture dishes (Nunc). At the end of coculture, the unbound sperm population was removed by washing each well five times as follows: 800 µl of fresh medium were added, and then the same volume was removed, after repeated suspensions, and discarded. Unbound sperm were removed from explants by transferring them three times in sp-TALP before observation. Oviductal monolayers (grown on glass coverslips) with attached sperm were fixed as described below.
Experiment 1 (n = 5) addressed the effect of different doses of heparin and fucoidan and of monolayer pretreatment with heparin and fucoidan on sperm binding. For this purpose, monolayers were preincubated for 10 min with sp-TALP alone or with heparin or fucoidan at 100, 10, 1, and 0.1 µg/ml in sp-TALP. Parallel wells pretreated with heparin or fucoidan at 1 mg/ml for 10 min were extensively washed with sp-TALP before insemination. Hoechst-labeled sperm were added at 0.5 x 106 motile sperm/well, and after 1 h of coculture, unbound sperm were removed and monolayers fixed and analyzed as described below.
Experiment 2 (n = 4) was performed to detect whether sperm pretreatment with heparin and fucoidan influenced sperm binding to oviductal monolayers. To this end, sperm recovered by Percoll centrifugation were divided into three aliquots that were preincubated for 5 min with heparin at 100 µg/ml, fucoidan at 10 µg/ml, or sp-TALP alone, then washed in sp-TALP by centrifugation at 200 x g for 10 min, and finally inseminated at 0.3 x 106 motile sperm/well in a final volume of 0.5 ml. The final concentrations of heparin and fucoidan in the wells inseminated with pretreated sperm were 0.2 and 0.04 µg/ml, respectively. Parallel control wells containing heparin at 0.2 µg/ml, fucoidan at 0.04 µg/ml, or sp-TALP alone were inseminated with the sperm aliquot that was pretreated in sp-TALP alone at the same concentration of motile sperm. Unbound sperm were removed 1 h after sperm addition, and monolayers were fixed for quantitative analysis.
Experiment 3 (n = 5) was designed to study the effect of pretreatment of oviductal monolayers with heparinase I, II, or sodium chlorate (an inhibitor of sulfation [29]) on sperm binding. Monolayers grown to confluence were exposed to heparinase I or II at 0.1 IU/ml in Dulbecco's PBS or buffer alone for 5 h at 35°C under 5% CO2 in air, washed with sp-TALP, and inseminated. Parallel wells, preincubated with or without 20 mM sodium chlorate in M199 with 10% w/v FCS at 38.5°C under 5% CO2 in air for 24 h, were washed with sp-TALP and inseminated at 0.8 x 106 motile sperm/well. Unbound sperm were removed 1 h after sperm addition, and monolayers were fixed for quantitative analysis.
Experiment 4 (n = 4) addressed the effect of different doses of heparin and fucoidan on sperm release. Wells were inseminated at 0.2 x 106 motile sperm/well in 0.5 ml of sp-TALP and washed free of unbound sperm at 1 h. Then, heparin or fucoidan was added at a final concentration of 0.1, 1, 10, or 100 µg/ml. Control as well as heparin- and fucoidan-treated wells were washed free of unbound sperm 1 h after the addition of sulfated polysaccharides and finally fixed for quantitative analysis.
Experiment 5 (n = 5) was designed to study the time required by a fixed dose of heparin or fucoidan to release sperm from oviductal monolayers. Heparin or fucoidan at 100 µg/ml were added to monolayers inseminated at 0.3 x 106 motile sperm/well and treated as described for experiment 4. Wells were washed free of unbound sperm and fixed at 5, 15, and 60 min after sperm addition.
Experiment 6 (n = 3) was designed to study whether inhibition of sperm binding or triggering of sperm release by the two sulfated glycoconjugates observed with oviductal monolayers related to the oviductal culture stage. For this purpose, oviductal explants (used within 24 h after collection) were inseminated at 0.25 x 106 motile sperm/ml with or without heparin or fucoidan at 100 µg/ml in 0.5 ml of sp-TALP, washed free of unbound sperm 1 h after sperm addition, and observed. The same concentrations of heparin and fucoidan were added to explants cocultured with sperm for 1 h in sp-TALP after elimination of unbound sperm. Removal of loosely bound sperm and fixation were performed 15 min after the addition of sulfated glycoconjugates.
Experiment 7 (n = 4) was designed to study the effect of heparin, N-desulfated heparin, dextran (MW of 500 000), and dextran sulfate (prepared from dextran with a MW of 500 000) on sperm binding and release from oviductal monolayers. For this purpose, oviductal monolayers were incubated with 0.3 x 106 motile sperm/well for 1 h with or without dextran, dextran sulfate, heparin, and N-desulfated heparin at 100 µg/ml. Then, they were washed free of unbound sperm, fixed, and observed for quantitative analysis. Parallel wells were added with polysaccharides at the same final concentration after 1 h of sperm interaction and removal of unbound sperm, incubated 15 min, washed free of unbound sperm, and fixed for quantitative analysis.
Quantitation of Number of Bound Sperm
Monolayers grown on coverslips and inseminated with Hoechst-labeled sperm were fixed in 2.5% w/v glutaraldheyde in PBS for 1 h at room temperature (20–25°C), extensively washed, and mounted with the same buffer on a glass slide with cells facing up. For each well, fields of 0.286 mm2 were acquired at a Zeiss Axioplan microscope equipped with phase-contrast, fluorescence, and Nomarsky optics by means of an Optronix camera and KS 300 software (Zeiss, Milan, Italy). Number of bound sperm was determined by analyzing 10 fields of 0.286 mm2 for each well. Experiments done on explants were only observational.
Statistical Analysis
Data were analyzed by one-way ANOVA (Systat 9; Spss, Inc., Milan, Italy) followed by pair-wise comparison of means with Tukey's honestly significant difference.
RESULTS
Experiment 1
In the present study, experiment 1 was performed to investigate the effects of different doses of heparin and fucoidan as well as of monolayer pretreatment with the two sulfated glycoconjugates on sperm binding. Sperm adhesion was markedly inhibited by heparin at 10 µg/ml and nearly abolished at 100 µg/ml (control vs. heparin at 0.1 µg/ml, P > 0.05; control vs. heparin at 1, 10, and 100 µg/ml, P < 0.001; n = 5; Fig. 1a). Interestingly, monolayer pretreatment with heparin at 1 mg/ml for 10 min, followed by extensive rinses before insemination in sp-TALP alone, as shown in Figure 1a, did not affect the number of bound sperm (control vs. heparin pretreatment, P > 0.05; n = 4). As regards fucoidan, sperm adhesion was markedly inhibited at 0.1 µg/ml and nearly abolished at 1 µg/ml (control vs. fucoidan at 0.1 and 1 µg/ml, P < 0.001; n = 5; Fig. 1b). As shown in Figure 1b, monolayer pretreatment with fucoidan at 1 mg/ml for 10 min, followed by insemination in fucoidan-free sp-TALP, had no effect on the number of bound sperm (control vs. fucoidan pretreatment, P > 0.05; n = 5).
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Experiment 2
Data from experiment 1 showed that fucoidan inhibited sperm binding from 10- to 50-fold more effectively than heparin. Therefore, sperm pretreatment was performed with sp-TALP plus 100 µg/ml of heparin, sp-TALP plus 10 µg/ml of fucoidan, or sp-TALP alone for 5 min. Data showed that sperm pretreatment with both sulfated glycoconjugates produced a significant inhibition compared to control in sp-TALP alone as well as compared to controls with the same final concentrations of the two polysaccharides (control vs. heparin or fucoidan pretreatment, P < 0.001; heparin pretreatment vs. heparin control and fucoidan pretreatment vs. fucoidan control, P < 0.001; n = 4; Fig. 2). Moreover, the concentration of heparin (0.2 µg/ml) present in the control well significantly inhibited sperm binding compared to control in sp-TALP alone (P < 0.05), whereas fucoidan (0.04 µg/ml) did not (P > 0.05, n = 4, Fig. 2).
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Experiment 3
The inhibitory effect of heparin might be due to the presence of heparin-like glycosaminoglycans on the apical surface of cultured oviductal cells. To test this hypothesis, the effects of oviductal cell treatment with heparinase I, II, and sodium chlorate on sperm binding was investigated. Data showed that neither heparinase I nor sodium chlorate had significant effects on sperm binding (P > 0.05, n = 4, Fig. 3), whereas heparinase II significantly increased the number of bound sperm compared to the control (P < 0.001, n = 4, Fig. 3).
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Experiment 4
Analysis of data regarding the effects of heparin and fucoidan on release of sperm bound to oviductal monolayers showed that heparin begins to induce a significant sperm release at 1 µg/ml that increases up to 96% at 100 µg/ml (P < 0.001, n = 4, Fig. 4a). As regards fucoidan, a significant release was observed at 0.1 µg/ml (67% of bound sperm) and at 1 µg/ml (91%), but no bound sperm were observed at 10 µg/ml (P < 0.001, n = 4, Fig. 4b).
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Experiment 5
Observation of living cocultures at the moment of sulfated glycoconjugate addition showed a sudden increase of flagellar beat frequencies, followed by a gradual release of sperm with high linear motility (Fig. 5). Quantitative analysis revealed that sperm release was nearly completed within 5 min after the addition of fucoidan or heparin at 100 µg/ml (P < 0.001, n = 5, Fig. 6).
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Experiment 6
To test the hypothesis that inhibition of binding or triggering of release by the two sulfated glycoconjugates observed with oviductal monolayers may be affected by the oviductal culture stage, oviductal explants were used. Explants inseminated in sp-TALP plus 100 µg/ml of heparin or fucoidan bound a negligible number of bound sperm compared to control. Moreover, addition of 100 µg/ml of heparin or fucoidan to parallel explants cocultured for 1 h induced a nearly complete release of bound sperm (data not shown).
Experiment 7
Results of the experiments described above point out that the sulfated glycoconjugates heparin and fucoidan are potent inhibitors of sperm binding and triggers of sperm release from in vitro cultured bovine oviductal monolayers. To test whether the sulfation of such molecules was responsible for the observed effects, heparin, N-desulfated heparin, dextran (MW of 500 000), and dextran sulfate (prepared from dextran with a MW of 500 000) were tested on sperm binding and release. Heparin and dextran sulfate, but not N-desulfated heparin and dextran, at 100 µg/ml exerted a marked inhibition on sperm adhesion and triggered an almost complete release of bound sperm (control vs. dextran sulfate or heparin, P < 0.001; n = 4; Fig. 7). As in the case of heparin and fucoidan, observation of living cocultures at the time of dextran sulfate addition showed a sudden increase of the flagellar beat frequency, followed by a gradual release of bound sperm during the first few minutes after addition.
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DISCUSSION
During the last 10 yr, several studies have shown that mammalian sperm transiently adhere to the epithelial cells lining the fallopian tube during their ascent in the female reproductive tract until unknown ovulatory signals release fertile sperm within the oviduct [9]. To investigate the details of sperm-oviduct interaction, studies with in vitro systems provided accumulating evidence about the role of adhesion and oviductal secretions in the maintenance of sperm life span in different species [4–8; 12–22]. However, little is known about the mechanisms involved in the release of fertile sperm. To our knowledge, the only available data demonstrate that sperm release is not due to changes of the oviductal mucosa [11, 23], and that capacitation reduces the ability of sperm to adhere to the oviductal epithelium [11, 30, 31]. On the basis of such indirect evidence, it has been suggested that sperm capacitation and hyperactivation, induced by unknown signals, may enhance the ability of sperm to release themselves [11, 31, 32]. In the present study, several glycoconjugates, which generally are used as capacitating agents during bovine in vitro fertilization [27, 33, 34], have been tested for their ability to affect sperm adhesion. We demonstrated, to our knowledge for the first time, that heparin, fucoidan, and other sulfated glycoconjugates, but not their unsulfated counterparts, at nanomolar to micromolar concentrations are powerful inhibitors of sperm binding to oviductal cells in vitro and, more interestingly, are able to trigger the mechanism of sperm release, probably through early capacitative effects. The overall results support the hypothesis that changes in glycoconjugate concentration during the estrous cycle drive the release of fertilization-competent sperm from the oviductal reservoir, allowing their ascension toward the ampulla.
The first results obtained in the present study demonstrate that heparin and fucoidan are powerful inhibitors of sperm binding to oviductal monolayers in a dose-dependent manner. Data from pretreatment experiments, showing that both glycoconjugates act on sperm rather than on oviductal epithelial cells, may indicate that exogenously added glycoconjugates may compete with similar molecules involved in sperm adhesion that are exposed on the surface of oviductal cells. However, findings from further experiments argue against this hypothesis. In fact, extensive digestion of monolayers with heparinase I or II before insemination had no effect on subsequent sperm adhesion. Moreover, because a structural similarity between the two glycoconjugates is represented by the presence of sulfate groups, which possibly are involved in the adhesion, oviductal monolayers were also treated with sodium chlorate, an inhibitor of sulfotransferases, before insemination. As in the case of heparinase digestion, sulfation inhibition did not affect sperm adhesion in vitro. These findings suggest the alternative hypothesis that sperm interaction with exogenously added glycoconjugates induce a remodeling of the sperm surface, causing the loss or modification of molecules actually involved in sperm-oviduct binding. Heparin is a well-known capacitating agent routinely used during bovine in vitro fertilization [27, 33, 35], but it is worth mentioning that the fucose sulfate glycoconjugate from the sea urchin jelly coat, which has a remarkable similarity with fucoidan, is also able to capacitate bovine sperm [34]. Moreover, preliminary in vitro fertilization experiments carried out in our laboratory demonstrate that fucoidan is also able to capacitate bovine sperm (unpublished results).
Recently, fucoidan, but not heparin, has been shown to inhibit sperm adhesion to bovine oviductal explants, and on the basis of this and other evidence, it has been suggested that fucose residues exposed on the apical surface of oviductal cells may mediate sperm-oviduct adhesion [26]. In the present study, oviductal monolayers were used instead of explants, because as previously shown [23], such a culture stage allows better bound sperm visualization and, consequently, more reliable counts. Moreover, the use of sperm labeled with Hoechst 33342 before insemination further improved sperm visualization in our adhesion assay. Under these conditions, fucoidan and heparin, at concentrations lower than those used by Lefebvre et al. [26], had a marked inhibitory effect on sperm adhesion. To understand whether discrepancies between our results as compared to those of Suarez [26] arise from differences in the oviductal culture stage, the effects of heparin and fucoidan on sperm adhesion were also assessed using oviductal explants. However, such discrepancies seem not to be related to the culture stage, because similar results were obtained using heparin and fucoidan at a final concentration of 100 µg/ml on oviductal explants.
The finding that sulfated glycoconjugates, which are able to capacitate sperm, induce a loss of sperm affinity for oviductal cells in vitro led us to test their effects directly on sperm release. The most striking finding in this study is that heparin and fucoidan are also able to release sperm bound to oviductal monolayers. The addition of such glycoconjugates to the cocultures caused a sudden increase of bound-sperm flagellar beating, followed by the release of sperm with high linear motilities during the first 5 min after addition. These novel findings may have several meanings and applications. First, they may provide new insights into the physiological mechanisms underlying sperm release within the fallopian tube, and second, a method to readily release sperm adhering to the tube in vitro is now available. In this regard, it is interesting to note that adhesion in vitro to the fallopian tube not only is beneficial for sperm fertile life, but it also allows the selection of higher-quality sperm. In fact, several reports in different species claim that the tube selects sperm characterized by intact acrosomes [23], uncapacitated status [11, 30, 31], superior morphology [36], and normal chromatin structure [37]. However, until now, this sperm population could not be recovered for further characterization or use in reproductive technologies, because sperm that are spontaneously and progressively released in the culture medium are mostly dead, have a limited motility [18], and have a high percentage of acrosome reactions [23]. Probably, such changes occur after release as a result of capacitation. The possibility of synchronously inducing the release of sperm bound to the bovine fallopian tube in vitro offers a unique opportunity for studying oviduct-selected, superior-quality sperm as well as for their utilization in reproductive biotechnologies.
As outlined above, addition of heparin or fucoidan causes release of the great majority of bound sperm during a very short period of time. If the release is due to the capacitating activity of such glycoconjugates, this means that some events of capacitation may occur very rapidly. Because other sulfated glycoconjugates, which generally are recognized as capacitating agents in the bovine fertilization, also caused sperm release, such an event may represent the earlier capacitative modification identified until now in bull sperm. In support of this hypothesis, sulfation seems to be essential for the capacitation effects of such glycoconjugates [34, 38] as well as for inducing the rapid loss of affinity of sperm for oviductal cells reported here. In fact, dextran and, in particular, N-desulfated heparin (the latter being known for its inability to capacitate bovine sperm [38]) do not modify sperm affinity for oviductal cells. Moreover, the addition of sulfated glycoconjugates first caused a sudden increase of bound sperm flagellar beat frequencies and only later brought about the release of sperm endowed with high linear motility. This finding suggests that sperm are able to recognize sulfated glycoconjugates, and that such an event is rapidly transduced across the sperm membrane, resulting in a marked increase of flagellar motility. Although capacitation is a central event in the life of mammalian sperm, the molecular mechanisms underlying such a biological process are still incompletely understood. Most studies on heparin-induced capacitation in the bovine species have dealt with capacitation endpoints, such as the ability to undergo the acrosome reaction or to fertilize an oocyte, that occur only after 4–6 h of incubation with heparin. Considerably less is known about the early events associated with heparin-induced capacitation. The finding that sperm loaded with the calcium indicator FURA increase their calcium ions concentration 60% over basal levels after 15 min of incubation with heparin [39], to our knowledge, is the only available data on early capacitation changes in the bovine species. In the present study, further early capacitative effects in the bovine sperm triggered by heparin and other sulfated glycoconjugates, such as inhibition of sperm binding, enhancement of motility of bound sperm, and subsequent induction of release, are demonstrated.
Also as outlined above, fucose residues exposed on the apical surface of oviductal cells have been suggested to mediate sperm-oviduct adhesion by binding a C-type lectin on the surface of acrosome-intact bull sperm [26, 40]. Sperm-oviduct binding inhibition in the presence of fucoidan was considered to be one of the evidences in support of this hypothesis [26]. However, evidence is also provided here that fucoidan, as other sulfated glycoconjugates, induces a loss of sperm affinity for oviductal cells through an early capacitative effect. Therefore, it can be hypothesized that molecules on the surface of bull sperm involved in the oviduct adhesion are different from those involved in the binding of sulfated glycoconjugates that mediate capacitation. Indeed, the possibility that sperm release does not rely on a loss of sperm affinity for the oviductal epithelium but, merely, on the increase of sperm flagellar beat frequencies induced by sulfated glycoconjugates cannot be ruled out on the basis of the present data. Whatever the case, the present results demonstrate, to our knowledge for the first time, that heparin and other sulfated glycoconjugates are powerful modulators of sperm-oviduct adhesion in vitro. Therefore, it can be suggested that glycosaminoglycans secreted by the oviduct at estrus also play a key role in sperm release from the oviductal reservoir in vivo. In this regard, it is worth noting that heparin-like glycosaminoglycans are present in the bovine oviductal fluid, and that their concentrations and capacitating activities change, being maximal at estrus [8]. Media conditioned by whole-oviduct segment [13] or monolayers [14, 15] also have a capacitating activity that peaks at estrus and declines during the luteal phase. Interestingly, the glycosaminoglycan concentration of media conditioned by whole-oviduct segment follows the same pattern [13]. In conclusion, we propose that sulfated glycosaminoglycans, secreted by the oviduct at estrus, progressively modulate sperm capacitation, first inducing sperm release from the oviductal reservoir and then enhancing sperm fertilization ability.
ACKNOWLEDGMENTS
We thank Mr. G. Falcone for printing.
FOOTNOTES
1 Supported by P.R.I.N. grant 1999 "Glicobiologia della fecondazione: struttura e funzione di glicoproteine di superficie di gameti e gamonti." 
2 Correspondence: R. Talevi, Dipartimento di Biologia Evolutiva e Comparata, Università di Napoli "Federico II," Via Mezzocannone 8, 80134 Napoli, Italy. FAX: 81 2528902;talevi{at}dgbm.unina.it
Accepted: September 12, 2000.
Received: June 13, 2000.
REFERENCES
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